Many industries, including pharmaceuticals, need to detect low levels of materials in large volumes of liquid. For example, they rely heavily on classical microbiological techniques to detect microbial contamination. Areas often under surveillance by Quality Assurance personnel include the control of bioburden in incoming raw materials, especially liquids; monitoring of microbial population in the production environment; in-process controls, especially after storage; and final product testing. Often product is manufactured and stored whilst analysis for microbial content takes place. If contamination is detected, the product may need to be destroyed, and the production line shut down until the source of contamination is found. Often the time taken for microbial analysis is the rate-limiting factor in bringing the plant on stream. This can lead to substantial costs in wasted production or raw materials.
The determination of the number of microorganisms present in the various types of water used in a pharmaceutical environment is considered a critical factor in producing many products. Usually, the microbiological specification ranges from 0.01 to 100 cfu per ml of water, depending on the source of the sample.
Classical methods of microbe detection, whilst considered reliable and accepted in the industry, are slow and require valuable storage and laboratory space.
A test for coliform bacilli is described in "Handbook of Practical Bacteriology" 9 (1956), by T. J. Mackie and J. E. McCartney, pub. E. and S. Livingstone. The method employs a specific medium which is utilised by coliforms to release acid, and thus to give a colour change with a pH indicator. Several tubes are set up, at different dilutions, and a McCrady table is used to determine the most probable number of bacilli, from the number of positives detected. This method allows the detection of small numbers of microbes in a large volume of liquid, but involves serial dilution and an incubation stage of 48 hours.
Cherwell Laboratories Ltd., Bicester, UK, and Wilkinson and Simpson Ltd., Gateshead, UK, each produce test kits, respectively under the trade names Colitrace and Colilert, designed to test water for levels of selected organisms down to 1 per 100 ml of sample. The sample is distributed between independent tubes containing culture broths, which are then incubated; the presence of coliforms is indicated by a colour change, and E. coli can be detected by fluorescence. The most probable number (MPN) test is then used to estimate numbers of organisms.
WO-A-8202561 and also Williams et al, Annals of the New York Acad. Sci. 501:350-353 (1987), disclose methods for the detection of microorganisms in gel microdroplets with counting, using a flow microfluorimeter. The technique is difficult to operate, for a large volume of sample containing a low number of microbes.
FR-A-2649411 discloses a method for quantitative determination of bacteria, by growing them on a semi-selective medium on a membrane support. The method is unsuitable for distinguishing between species of microorganisms.
GB-A-2035372 discloses a method for quantifying coliform bacilli. In an example, a 5 ml sample is measured at a maximum rate of 0.2 ml/min. This is too slow for large sample volumes.
Pat. Abs. Japan 9(299) (P-408)(2022) (Nov. 27, 1985), equivalent to JP-A-60135862, describes a method in which a serum sample is distributed over 128 wells, and growth medium is then added. The serum titer is assayed by then counting the number of wells which are closed by cell colonies. This technique is not suitable for assaying large sample volumes.
U.S. Pat. No. 3,929,583, and subsequent literature naming A. N. Sharpe as an author, describe membrane filters for enumerating microorganisms. The then novel membrane has a grid pattern imprinted on it, providing as many as 1600 "cells" in which microorganisms can be retained by filtration and grown to form colonies. Each cell has an area smaller than the normal colony size so that, after incubation, each existent colony is uniform in size and shape, and has high visibility. It can then be visualised, and enumerated by the MPN technique. This apparatus is shown to be preferable to conventional membrane filters, in that the latter did not allow a count higher than 400 cfu.
It is desirable, not only to enumerate total units of material in a sample, but also to identify specific types of material. For example, certain strains constituting the species Escherichia coli are capable of inducing disease. E. coli is therefore regarded as a potential pathogenic organism. Several different groups of diarrhoea-inducing strains are known. The enterotoxigenic E. Coli (ETEC) strains produce one or more toxins from the heat-labile and the heat-stable (ST) enterotoxin families. Traditionally, the ST enterotoxin has been detected by means of the infant mouse assay; a less costly and less time-consuming assay is desirable, which does not require animal facilities.
Multi-well plates are widely used in analytical laboratories. Some such plates have a filter material at the base of each well. This may be used to draw liquid through the wells under suction, after (different) samples have been introduced into each well.